This proposal is a competing renewal application of R01 CA074305 that combines the use of chemical approaches, enzymologic measurements, cellular analysis, and proteomics to enhance our understanding of protein kinases and phosphorylation in signaling. It is now well-accepted that protein kinases through protein phosphorylation can drive a broad range of cellular growth, differentiation, and motility pathways in normal and disease processes including cancer. Although our understanding of the mechanisms and functions of kinases and their regulation has increased dramatically over the past few decades, there are still large gaps in our understanding of how they drive particular biological and pathological outputs. Filling these knowledge gaps has the potential to provide a clearer understanding of basic biomedical processes and has the opportunity to enhance the development of novel therapeutic approaches and disease diagnostic strategies. There are three Specific Aims. 1. Clarify the mechanisms of oncogenic mutation and protein interactions in EGFR (Epidermal Growth Factor Receptor) tyrosine kinase signaling. We will use our newly developed approach to generate near full-length EGFR mutant proteins to understand their enzymologic features, inhibitor sensitivities, and interactions with the tumor suppressor Mig6. 2. Elucidate the molecular mechanisms of regulation of the tumor suppressor PTEN by C-terminal phosphorylation. Using expressed protein ligation, we will build on preliminary data to generate and analyze phosphorylated forms of PTEN with respect to understanding their altered catalytic activity and conformation. 3. Illuminate the regulatory roles of acetylation of protein kinases CK2 and protein kinase A. We will employ a newly developed method to install methylthiocarbamyl (MTC) acetyl-Lys mimics by Cys alkylation and analyze the biochemical effects. Taken together, we believe these aims have the potential to enhance our understanding of how key signaling molecules are regulated by mutation or post-translational modification and identify new therapeutic opportunities for cancer and other diseases. PUBLIC HEALTH RELEVANCE: This proposal promises to clarify important mechanistic features of proteins that play major roles in cancer and other diseases. It employs emerging chemical technologies along with cellular and biophysical methods to fill the gaps in our knowledge about cancer signaling networks. New insights from these studies may help shape the discovery and application of the next generation of anti-cancer drugs and diagnostic strategies.